P2Y4 antibody and methods of use

ABSTRACT

This invention provides a receptor having a preference for pyrimidine nucleotides preferably uridine triphosphate over purine nucleotides. A receptor having a preference for pyrimidine nucleotides over purine nucleotides means a receptor for which pyrimidine nucleotides and purine nucleotides are not equally active and equipotent. This means that the receptor according to the invention in presence of these agonists presents a functional response (preferably the accumulation of Inositol triphosphate (IP3), diacylglycerol (DAG), or calcium ions) to lower concentration of pyrimidine nucleotides, preferably uridine triphosphate, than to purine nucleotides or a more important functional response to similar concentration of pyrimidine nucleotide than to purine nucleotide

RELATED APPLICATIONS

[0001] This application is a divisional of application Ser. No.10/753,695, filed January 8, 2004, which is a divisional of applicationSer. No. 09/077,173, filed Nov. 12, 1998 which is the U.S. Nationalstage of International Application No. PCT/BE96/00123, filed on Nov. 21,1996, which designated the United States and was published in English,which claims priority to foreign application EP 95870124.5 filed on Nov.21, 1995. The entire teachings of the above application(s) areincorporated herein by reference.

OBJECT OF THE PRESENT INVENTION

[0002] The present invention concerns a new receptor having a preferencefor pyrimidine nucleotides preferably uridine triphosphate over purinenucleotides and the nucleic acid molecule encoding said receptor,vectors comprising said nucleic acid molecule, cells transformed by saidvector, antibodies directed against said receptor, nucleic acid probesdirected against said nucleic acid molecule, pharmaceutical compositionscomprising said produces and non human transgenic animals expressing thereceptor according to the invention or the nucleic acid moleculeaccording to said receptor.

[0003] The invention further provides methods for determining ligandbinding, detecting expression, screening for drugs, molecular bindingspecifically to said receptor and treatment involving the receptoraccording to the invention.

BACKGROUND

[0004] The cloning of several receptors for ATP has been reported since1993. In keeping with the latest nomenclature proposal, these P2purinergic receptors can be subdivided into two classes: Gprotein-coupled receptors, or P2Y receptors, and receptors withintrinsic ion channel activity or P2X receptors (2). Two distinct ratP2X receptors have been cloned, respectively from the vas deferens (3)and phaechromocytoma PC12 cells (4): they have a characteristictopology, with two hydrophobic putatively membrane-spanning segments andan ion pore motif reminiscent of potassium channels. In the P2Y family,the sequences of two subtypes, both coupled to phospholipase C, havebeen published: chick (5), turkey (6), bovine (7), mouse and rat (8)P2Y1 receptors (formerly called P2Y); murine (9, 10), rat (11) and human(12) P2Y2 receptors (previously named P2U) on the other hand. Inaddition, a P2Y3 receptor, with a preference for ADP over ATP, has beencloned from chick brain, but its sequence is not yet published (13).Furthermore, the 6H1 orphan receptor, cloned from activated chicken Tlymphocytes, exhibits a significant degree of homology to the P2Y1 andP2Y2 receptors, suggesting that it also belongs to the P2Y family,although its responsiveness to nucleotides has not yet been demonstrated(14).

SUMMARY OF THE INVENTION

[0005] This invention provides a receptor having a preference forpyrimidine nucleotides preferably uridine triphosphate over purinenucleotides. A receptor having a preference for pyrimidine nucleotidesover purine nucleotides means a receptor for which pyrimidinenucleotides and purine nucleotides are not equally active andequipotent. This means that the receptor according to the invention inpresence of these agonists presents a functional response (preferablythe accumulation of Inositol triphosphate (IP3), diacylglycerol (DAG),or calcium ions) to lower concentration of pyrimidine nucleotides,preferably uridine triphosphate, than to purine nucleotides or a moreimportant functional response to similar concentration of pyrimidinenucleotide than to purine nucleotide.

[0006] The inositol phosphate (IP3) accumulation after addition of saidagonists is described in the specification thereafter.

[0007] Advantageously, the receptor according to the invention has atleast a twofold, preferably a tenfold to one hundredfold preference forpyrimidine nucleotides over purine nucleotides.

[0008] A preferred embodiment of the receptor according to the inventionis characterized by a preference for uridine triphosphate over adeninenucleotides.

[0009] The receptor according to the invention is a receptor, preferablya G protein-coupled receptor, which belongs structurally to thepurinergic receptor family (P2Y family) but functionally is apyrimidinergic receptor, preferably a UTP-specific receptor.

[0010] According to a preferred embodiment of the present invention, thereceptor is a human receptor.

[0011] Said receptor has an amino acid sequence having more than 60%homology with the amino acid sequence shown in FIG. 1. Preferably, theamino acid sequence of the receptor according to the invention has atleast the amino acid sequence shown in FIG. 1 or a portion thereof.

[0012] A portion of the amino acid sequence means a peptide or a proteinhaving the same binding properties as the receptor according to theinvention (i.e. peptide or a protein which is characterized by apreference for pyrimidine nucleotides, preferably UTP, over purinenucleotides).

[0013] The present invention is also related to a nucleic acid molecule,such as a DNA molecule or an RNA molecule, encoding the receptoraccording to the invention.

[0014] Preferably, said DNA molecule is a cDNA molecule or a genomic DNAmolecule.

[0015] Preferably, the nucleic acid molecule according to the inventionis at least the DNA sequence shown in FIG. 1 or portion thereof. “Aportion of a nucleic acid sequence” means a nucleic acid sequenceencoding at least a portion of amino acid sequence as described above.

[0016] The present invention is also related to a vector comprising thenucleic acid molecule according to the invention. Preferably, saidvector is adapted for expression in a cell and comprises the regulatoryelements necessary for expressing the amino acid molecule in said celloperatively linked to the nucleic acid sequence according to theinvention as to permit expression thereof.

[0017] Preferably, said cell is chosen among the group consisting ofbacterial cells, yeast cells, insect cells or mammalian cells. Thevector according to the invention is a plasmid or a virus, preferably abaculovirus, an adenovirus or a semliki forest virus.

[0018] The plasmid may be the pcDNA3-P2Y4.

[0019] The present invention concerns also the cell (preferably amammalian cell, such as a 1321N1 cell) transformed by the vectoraccording to the invention. Advantageously, said cell is preferably nonneuronal in origin and is chosen among the group consisting of a COS-7cell, an LM(tk-) cell, an NIH-3T3 cell or a 1321N1 cell.

[0020] The present invention is also related to a nucleic acid probecomprising the nucleic acid molecule according to the invention, of atleast 15 nucleotides capable of specifically hybridizing with a uniquesequence included in the sequence of the nucleic acid molecule encodingthe receptor according to the invention. Said nucleic acid probe may bea DNA or an RNA molecule.

[0021] The invention concerns also an antisense oligonucleotide having asequence capable of specifically hybridizing to an mRNA moleculeencoding the receptor according to the invention so as to preventtranslation of said mRNA molecule or an antisense oligonucleotide havinga sequence capable of specifically hybridizing to the cDNA moleculeencoding the receptor according to the invention.

[0022] Said antisense oligonucleotide may comprise chemical analogs ofnucleotide or substances which inactivate mRNA, or be included in an RNAmolecule endowed with ribozyne activity.

[0023] Another aspect of the present invention concerns a ligand otherthan purine and pyrimidine nucleotides (preferably an antibody) capableof binding to a receptor according to the invention and an anti-ligand(preferably also an antibody) capable of competitively inhibiting thebinding of said ligand to the receptor according to the invention.

[0024] Preferably, said antibody is a monoclonal antibody.

[0025] The present invention concerns also the monoclonal antibodydirected to an epitope of the receptor according to the invention andpresent on the surface of a cell expressing said receptor.

[0026] The invention concerns also the pharmaceutical compositioncomprising an effective amount of oligonucleotide according to theinvention, effective to decrease the activity of said receptor bypassing through a cell membrane and binding specifically with mRNAencoding the receptor according to the invention in the cell so as toprevent its translation. The pharmaceutical composition comprises also apharmaceutically acceptable carrier capable of passing through said cellmembrane.

[0027] Preferably, in said pharmaceutical composition, theoligonucleotide is coupled to a substance, such as a ribozyme, whichinactivates mRNA.

[0028] Preferably, the pharmaceutically acceptable carrier comprises astructure which binds to a receptor on a cell capable of being taken upby cell after binding to the structure. The structure of thepharmaceutically acceptable carrier in said pharmaceutical compositionis capable of binding to a receptor which is specific for a selectedcell type.

[0029] Preferably, said pharmaceutical composition comprises an amountof the antibody according to the invention effective to block thebinding of a ligand to the receptor according to the invention and apharmaceutically acceptable carrier.

[0030] The present invention concerns also a transgenic non human mammaloverexpressing (or expressing ectopically) the nucleic acid moleculeencoding the receptor according to the invention.

[0031] The present invention also concerns a transgenic non human mammalcomprising a homologous recombination knockout of the native receptoraccording to the invention.

[0032] According to a preferred embodiment of the invention, thetransgenic non human mammal whose genome comprises antisense nucleicacid complementary to the nucleic acid according to the invention is soplaced as to be transcripted into antisense MRNA which is complementaryto the MRNA encoding the receptor according to the invention and whichhybridizes to mRNA encoding said receptor, thereby reducing itstranslation. Preferably, the transgenic non human mammal according tothe invention comprises a nucleic acid molecule encoding the receptoraccording to the invention and comprises additionally an induciblepromoter or a tissue specific regulatory element.

[0033] Preferably, the transgenic non human mammal is a mouse.

[0034] The invention relates to a method for determining whether aligand can be specifically bound to the receptor according to theinvention, which comprises contacting a cell transfected with a vectorexpressing the nucleic acid molecule encoding said receptor with theligand under conditions permitting binding of ligand to such receptorand detecting the presence of any such ligand bound specifically to saidreceptor, thereby determining whether the ligand binds specifically tosaid receptor.

[0035] The invention relates to a method for determining whether aligand can specifically bind to a receptor according to the invention,which comprises preparing a cell extract from cells transfected with avector expressing the nucleic acid molecule encoding said receptor,isolating a membrane fraction from the cell extract, contacting theligand with the membrane fraction under conditions permitting binding ofthe ligand to such receptor and detecting the presence of any ligandbound to said receptor, thereby determining whether the compound iscapable of specifically binding to said receptor. Preferably, saidmethod is used when the ligand is not previously known.

[0036] The invention relates to a method for determining whether aligand is an agonist of the receptor according to the invention, whichcomprises contacting a cell transfected with a vector expressing thenucleic acid molecule encoding said receptor with the ligand underconditions permitting the activation of a functional receptor responsefrom the cell and detecting by means of a bio-assay, such as amodification in a second messenger concentration or a modification inthe cellular metabolism (preferably determined by the acidification rateof the culture medium), an increase in the receptor activity, therebydetermining whether the ligand is a receptor agonist.

[0037] The invention relates to a method for determining whether aligand is an agonist of the receptor according to the invention, whichcomprises preparing a cell extract from cells transfected with a vectorexpressing the nucleic acid molecule encoding said receptor, isolating amembrane fraction from the cell extract, contacting the membranefraction with the ligand under conditions permitting the activation of afunctional receptor response and detecting by means of a bio-assay, suchas a modification in the production of a second messenger an increase inthe receptor activity, thereby determining whether the ligand is areceptor agonist.

[0038] The present invention relates to a method for determining whethera ligand is an antagonist of the receptor according to the invention,which comprises contacting a cell transfected with a vector expressingthe nucleic acid molecule encoding said receptor with the ligand in thepresence of a known receptor agonist, under conditions permitting theactivation of a functional receptor response and detecting by means of abio-assay, such as a modification in second messenger concentration or amodification in the cellular metabolism, (preferably determined by theacidification rate of the culture medium) a decrease in the receptoractivity, thereby determining whether the ligand is a receptorantagonist.

[0039] The present invention relates to a method for determining whethera ligand is an antagonist of the receptor according to the invention,which comprises preparing a cell extract from cells transfected with anexpressing the nucleic acid molecule encoding said receptor, isolating amembrane fraction from the cell extract, contacting the membranefraction with the ligand in the presence of a known receptor agonist,under conditions permitting the activation of a functional receptorresponse and detecting by means of a bio-assay, such as a modificationin the production of a second messenger, a decrease in the receptoractivity, thereby determining whether the ligand is a receptorantagonist.

[0040] Preferably, the second messenger assay comprises measurement ofintracellular cAMP, intracellular inositol phosphate (IP3),intracellular diacylglycerol (DAG) concentration or intracellularcalcium mobilization.

[0041] Preferably, the cell used in said method is a mammalian cell nonneuronal in origin, such as a COS-7 cell, a CHO cell, a LM(tk-) cell anNIH-3T3 cell or 1321N1.

[0042] In said method, the ligand is not previously known.

[0043] The invention is also related to the ligand isolated and detectedby any of the preceding methods.

[0044] The present invention concerns also the pharmaceuticalcomposition which comprises an effective amount of an agonist or anantagonist of the receptor according to the invention, effective toreduce the activity of said receptor and a pharmaceutically acceptablecarrier.

[0045] For instance, said agonist or antagonist may be used in apharmaceutical composition in the treatment of cystic fibrosis, and themethod according to the invention may be advantageously used in thedetection of improved drugs which are used in the thereby determiningwhether the ligand is a receptor antagonist.

[0046] The present invention relates to a method for determining whethera ligand is an antagonist of the receptor according to the invention,which comprises preparing a cell extract from cells transfected with anexpressing the nucleic acid molecule encoding said receptor, isolating amembrane fraction from the cell extract, contacting the membranefraction with the ligand in the presence of a known receptor agonist,under conditions permitting the activation of a functional receptorresponse and detecting by means of a bio-assay, such as a modificationin the production of a second messenger, a decrease in the receptoractivity, thereby determining whether the ligand is a receptorantagonist.

[0047] Preferably, the second messenger assay comprises measurement ofintracellular cAMP, intracellular inositol phosphate (IP3),intracellular diacylglycerol (DAG) concentration or intracellularcalcium mobilization.

[0048] Preferably, the cell used in said method is a mammalian cell nonneuronal in origin, such as a COS-7 cell, a CHO cell, a LM(tk-) cell anNIH-3T3 cell or 1321N1.

[0049] In said method, the ligand is not previously known.

[0050] The invention is also related to the ligand isolated and detectedby any of the preceding methods.

[0051] The present invention concerns also the pharmaceuticalcomposition which comprises an effective amount of an agonist or anantagonist of the receptor according to the invention, effective toreduce the activity of said receptor and a pharmaceutically acceptablecarrier.

[0052] For instance, said agonist or antagonist may be used in apharmaceutical composition in the treatment of cystic fibrosis, and themethod according to the invention may be advantageously used in thedetection of improved drugs which are used in the treatment of cysticfibrosis.

[0053] Therefore, the previously described methods may be used for thescreening of drugs to identify drugs which specifically bind to thereceptor according to the invention.

[0054] The invention is also related to the drugs isolated and detectedby any of these methods.

[0055] The present invention concerns also a pharmaceutical compositioncomprising said drugs and a pharmaceutically acceptable carrier.

[0056] The invention is also related to a method of detecting expressionof a receptor according to the invention by detecting the presence ofMRNA coding for a receptor, which comprises obtaining total RNA or totalmRNA from the cell and contacting the RNA or MRNA so obtained with thenucleic acid probe according to the invention under hybridizingconditions and detecting the presence of mRNA hybridized to the probe,thereby detecting the expression of the receptor by the cell.

[0057] Said hybridization conditions are stringent conditions.

[0058] The present invention concerns also the use of the pharmaceuticalcomposition according to the invention for the treatment and/orprevention of cystic fibrosis.

[0059] The present invention concerns also a method for diagnosing apredisposition to a disorder associated with the activity of thereceptor according to the invention. Said method comprises:

[0060] a) obtaining nucleic acid molecules of subjects suffering fromsaid disorder;

[0061] b) performing a restriction digest of said nucleic acid moleculeswith a panel of restriction enzymes;

[0062] c) electrophoretically separating the resulting nucleic acidfragments on a sized gel;

[0063] d) contacting the resulting gel with a nucleic acid probe capableof specifically hybridizing to said nucleic acid molecule and labeledwith a detectable marker;

[0064] e) detecting labeled bands which have hybridized to the saidnucleic acid molecule labeled with a detectable marker to create aunique band pattern specific to subjects suffering from said disorder;

[0065] f) preparing nucleic acid molecules obtained for diagnosis bystep a-e; and

[0066] g) comparing the unique band pattern specific to the nucleic acidmolecule of subjects suffering from the disorder from step e and thenucleic acid molecule obtained for diagnosis from step f to determinewhether the patterns are the same or different and to diagnose therebypredisposition to the disorder if the patterns are the same.

[0067] A last aspect of the present invention concerns a method ofpreparing the receptor according to the invention, which comprises:

[0068] a) constructing a vector adapted for expression in a cell whichcomprises the regulatory elements necessary for the expression ofnucleic acid molecules in the cell operatively linked to nucleic acidmolecule encoding said receptor so as to permit expression thereof,wherein the cell is selected from the group consisting of bacterialcells, yeast cells, insect cells and mammalian cells;

[0069] b) inserting the vector of step a in a suitable host cell;

[0070] c) incubating the cell of step b under conditions allowing theexpression of the receptor according to the invention;

[0071] d) recovering the receptor so obtained; and

[0072] e) purifying the receptor so recovered, thereby preparing anisolated receptor according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0073]FIG. 1 The putative membrane-spanning domains are underlined andnumbered I to VII. The consensus sequence conserved between all the P2Yreceptors and the three amino acids (AHN) corresponding to the RGDsequence in the first extracellular loop of the P2Y₂ receptor arerepresented in bold. The putative phosphorylation sites by PKC or bycalmodulin-dependent protein kinases and PKC are indicated respectivelyby black squares (▪) and by open circles (∘).

[0074]FIG. 2 is a dendrogram representing structural relatedness amongthe cloned P2Y receptor and the closest neighbour in the Gprotein-coupled receptor family. The plot was constructed using themultiple sequence alignment program Pileup of the GCG package (26). Foreach sequence, the analysis takes into account a segment covering thefirst five putative membrane-spanning domains.

[0075]FIG. 3 represents a northern blot analysis of P2Y₄ receptorexpression. The Northern blot was performed with 15 μg of total RNA fromhuman placenta and 4 μg of poly(A)⁺RNA from K562 cells and from twodifferent human placentas. The probe was a human P2Y₄ gene fragmentamplified by PCR (TM2 to TM7).

[0076]FIG. 4 represents the time course of InsP₃ accumulation in 1321N1cells expressing the human P2Y₄ receptor. ³H inositol labelled cellswere incubated for the indicated time with UTP (100 μM), UDP (100 μM)and ATP (100 μM) in the absence of 10 mM LiCl (panel A) or in itspresence (panel B). The data represent the mean of triplicateexperimental points and are representative of two independentexperiments.

[0077]FIG. 5 Represents the effect of ATP on the accumulation of InsP₃induced by UTP in 1321N1 transfected cells. Concentration-action curvesof ATP in the presence of UTP 10 or 100 μM at 30 s (panel A) and 20 min(panel B). Concentration-action curve of ATP with or without UTP (10 μM)at 20 min (panel C). The data represent the mean±S.D. of triplicateexperimental points and are representative of two (panel A), five (panelB) or three (panel C) independent experiments.

[0078]FIG. 6 represents the concentration-action curves of UTP and UDPon the InsP₃ accumulation in three different clones of 1321N1transfected cells. The cells were incubated in the presence of variousUTP () and UDP (▪) concentrations (0, 0.1, 1, 3, 10 and 100 μM) for 30s or 20 min. The data represent the mean±S.D. of triplicate experimentalpoints obtained in one representative experiment. The EC₅₀ values weredetermined by curve fitting (Sigma Plot: version 2.0).

[0079]FIG. 7 Represents the effect of various nucleotides on the InsP₃production in 1321N1 transfected cells.

[0080] The cells were incubated with UTP, UDP, 5BrUTP, dUTP, ITP, AP₃A,AP₄A, AP₅A and AP₆A at the same concentration of 100 μM or withoutagonist (Cont) for 30 s or 20 min. The data represent the mean±S.D. oftriplicate experimental points and are representative of threeindependent experiments. The EC₅₀ values were determined by curvefitting (Sigma Plot: version 2.0).

[0081]FIG. 8 Represents concentration-action curves of variousnucleotides on the InsP₃ accumulation in 1321N1 cells expressing a humanP2Y₄ receptor. 1321N1 cells were incubated in the presence of variousconcentrations of UTP, UDP, dUTP, 5BrUTP, ITP and ATP for a period oftime of 20 min. The data are the mean±arange of duplicate experimentalpoints obtained in an experiment representative of two.

[0082]FIG. 9 Represents the action of various P₂ antagonists on theInsP₃ production induced by UTP in 1321N1 transfected cells. Cells wereincubated in the presence of suramin, reactive blue 2 and PPADS at aconcentration of 100 μM and different UTP concentrations (0, 2 and 10μM) for 20 min. The data represent the mean±S.D. of triplicateexperimental points and are representative of two independentexperiments.

[0083]FIG. 10 Represents the effect of PPADS on the UTP stimulation ofInsP₃ in 1321N1 transfected cells. The cells were exposed to variousconcentrations of UTP in the presence or in the absence of PPADS (100μM) for 20 min. The data are the mean±S.D. of triplicate experimentalpoints obtained in an experiment representative of two.

[0084]FIG. 11 Represents the effect of pertussis toxin on theUTP-induced accumulation of InsP₃ in 1321N1 cells expressing a humanP2Y₄ receptor. The cells were preincubated for 18 hours in the presenceor in the absence of 20 ng/ml pertussis toxin. The cells were thenincubated with or without UTP 100 μM and with or without pertussis toxin(20 ng/ml) for various times: 30 s, 5 min or 20 min. The data representthe mean±S.D. of triplicate experimental points and are representativeof two independent experiments.

DETAILED DESCRIPTION OF THE INVENTION EXPERIMENTAL PROCEDURES

[0085] 1. Materials

[0086] Trypsin was from Flow Laboratories (Bioggio, Switzerland) and theculture media, reagents, G418, fetal calf serum (FCS), restrictionenzymes and Taq polymerase were purchased from GIBCO BRL (Grand Island,N.Y.). The radioactive products myo-D-[2-³H]inositol (17.7 Ci/mmol) and[a³²P]ATP (800 Ci/mmol) were from Amersham (Gent, Belgium). Dowex AG1X8(formate form) was from Bio-Rad Laboratories (Richmond, Calif.). UTP,UDP, ATP, ADP, carbachol, LiCi and apyrase grade VII were obtained fromSigma Chemical Co. (St. Louis, Mo.). 2MeSATP was from ResearchBiochemicals Inc. (Natick, Mass.). pcDNA3 is an expression vectordeveloped by Invitrogen (San Diego, Calif.).

[0087] 2. Cloning and Sequencing

[0088] Degenerate oligonucleotide primers were synthesized on the basisof the best conserved segments between the murine P2Y2 and the chickP2Y1 receptor sequences. These primers were used to amplify novelreceptor gene fragments by low-stringency PCR starting from humangenomic DNA. The amplification conditions were as follows: 93° C. 1 min,50° C. 2 min, 72° C. 3 min; 35 cycles. The PCR products with sizescompatible with P2 receptor gene fragments were subcloned in M13mpl8 andM13mp19 and sequenced by the Sanger dideoxy nucleotide chain terminationmethod. One of the resulting clones sharing similarities with P2receptors, was labeled by random priming and used to screen a humangenomic DNA library constructed in the λ Charon 4a vector. Thehybridization was in 6×SSC (1×SSC: 0.15 M NaCl, 0.015 M Sodium citrate)and 40% formamide at 42° C. for 14 h and the final wash conditions were0.1×SSC, 0.1% SDS at 65° C. A preparation of λ phages (15) was made forseveral clones which hybridized strongly with the probe. A restrictionmap and a Southern blotting analysis allowed to isolate a 1.4 kbNheI-EcoRV fragment that was subcloned into the pBluescript SK³¹ vector(Stratagene). The complete sequence of a new receptor coding sequencewas obtained on both strands after subcloning of overlapping fragmentsin M13mp18 and M13mp19.

[0089] 3. Cell Culture and Transfection

[0090] The P2Y₄ receptor coding sequence was subcloned between theHindIII and the EcoRV sites of the pcDNA3 expression vector fortransfection into 1321N1 human astrocytoma cells, a cell line which doesnot respond to nucleotides and which has already been used for theexpression of purinergic receptors (6, 12). Cells were transfected withthe recombinant pcDNA3 plasmid (pcDNA3-P2Y₄) using the calcium phosphateprecipitation method as described (16). 1321N1 cells were incubated for6 hours at 37° C. in the presence of pcDNA3 vector alone or vectorcontaining the P2Y₄ receptor coding sequence, then washed and incubatedin culture medium (10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycinand 2.5 μg/ml amphotericin B in Dulbecco's modified Eagle's medium(DMEM)). The selection with G418 (400 μg/ml) was started two days aftertransfection. From the pool of transfected 1321N1 cells, individualclones were isolated by limiting dilution with the aim of selectingclones with high IP stimulation factors in response to nucleotides. Thedifferent clones were maintained in a medium containing 400 μg/ml G418.

[0091] 4. Inositol Phosphates (IP) Measurement

[0092] 1321N1 cells were labeled for 24 hours with 10 μCi/ml [³H]inositol in inositol-free DMEM (Dulbecco's modified Eagle's medium)medium containing 5% fetal calf serum, 100 U/ml penicillin, 100 μg/mlstreptomycin, 2.5 μg/ml amphotericin B and 400 pg/ml G418. Cells werewashed twice with KRH (Krebs-Ringer Hepes) buffer of the followingcomposition: (124 mM NaCl, 5 mM KCl, 1.25 mM MgSC₄, 1.45 mM CaCl₂, 25 mMHepes (pH 7.4) and 8 mM glucose) and incubated in this medium for 30min. The agonists were added in the presence of LiCl (10 mM) and theincubation was stopped after 30 s, 5 min or 20 min by the addition of anice-cold 3% perchloric acid solution. For the time course study, LiCl(10 mM) was added 5 min before the agonists and the incubation wasstopped at different times. When tested, pertussis toxin (20 ng/ml) wasadded for 18 h during the labeling period time and during thestimulation by the agonist. Inositol phosphates were extracted and InsP₃was isolated by chromatography on Dowex column as described previously(17).

[0093] 5. Radioligand Binding Assay.

[0094] Binding assays of [α³²P] UTP to cell membranes were carried outin Tris-HCl (50 mM, pH 7.5), EDTA 1 mM in a final volume of 0.5 ml,containing 25-50 μg of protein and 0.5 nM of radioligand (27). Theassays were conducted at 30° C. for 5 min. Incubations were stopped bythe addition of 4 ml of ice-cold Tris-HCl (50 mM, pH 7.5) and rapidfiltration through Whatman GF/B filters under reduced pressure. Thefilters were then washed three times with 2 ml of the same ice-coldTris-HCl buffer. Radioactivity was quantified by liquid scintillationcounting, after an overnight incubation of the filters in liquidscintillation mixture.

[0095] 6. Northern Blot and Southern Blot Analysis

[0096] Total and poly(A)+RNA were prepared from different tissues andhuman cell lines using the guanidinium thiocyanate-cesium chlorideprocedure (15), denatured by glyoxal and fractionated by electrophoresison a 1% agarose gel in 10 mM phosphate buffer pH 7.0. DNA samples,prepared from the λ Charon 4a clones, were digested with restrictionenzymes. Northern and Southern blots were prepared (15) and baked for 90min at 80° C. Membranes were prehybridized for at least 4 hours andhybridized overnight with the same probe as for the screening, at 42° C.in a solution containing 50% formamide for Northern blots and 40%formamide for Southern blots. Filters were washed twice for 15 min in2×SSC at room temperature and then twice for 30 min in 0.2×SSC at 60° C.before being exposed at −70° C. in the presence of intensifying screensfor 5 days (Northern blots) or 1 hour (Southern blots).

[0097] Results

[0098] 1. Cloning and Sequencing

[0099] In order to isolate new subtypes of P2 receptors, sets ofdegenerate oligonucleotide primers were synthesized on the basis of thebest conserved segments in the published sequences of the chick brainP2Y1 (5) and murine neuroblastoma P2Y2 (9) receptors. These primers wereused in low-stringency PCR on human genomic DNA as described (18). Somecombinations generated discrete bands with a size compatible with thatexpected for P2 receptors. For example, the primer5′CAGATCTAGATA(CT)ATGTT(CT)(AC)A(CT)(CT)T(ACGT) GC-3 corresponding tothe second transmembrane region and the primer5′-TCTTAAGCTTGG(AG)TC(ACG-T)A(CG)(AG)CA(AG)CT(AG) TT-3′ corresponding tothe seventh transmembrane region amplified a 712 bp fragment. Thepartial sequences obtained after sequencing were translated intopeptidic sequences and compared to a local databank which contains Gprotein-coupled receptor sequences. Most of the clones resulting fromthese PCR products encoded a part of a new receptor which displayed 58%identity with the murine P2Y2 receptor and 42% identity with the chickP2Y1 receptor partial sequences. In addition, some clones encoded apeptidic sequence presenting 87% identity with the chick P2Y1 receptorand are therefore believed to represent fragments of the human P2Y1gene.

[0100] The partial sequence of the new receptor was used as a probe toscreen a human genomic DNA library. Several clones that stronglyhybridized with the probe at high stringency conditions were obtainedand purified. The inserts of the clones varied from 12 to 17 kb andrestriction analysis revealed that all clones belonged to a singlelocus. The full sequence of a 1.4 kb NheI-EcoRV fragment was obtainedand an intronless open reading frame of 1095 bp was identified. Thesequence is depicted in FIG. 1 where the putative membrane-spanningdomains are underlined and numbered I to VII. The predicted molecularweight of the encoded protein is 36.5 kDa. This molecular weight isunlikely to be modified in vivo, since no N-glycosylation consensussequences are found in the putative exofacial regions. In contrast withthe human P2Y2 receptor, there is no RGD motif, an integrin bindingconsensus sequence, in the putative first extracellular loop. The threeamino acid (AHN) corresponding to the RGD sequence in the firstextracellular loop of the P2Y2 receptor are represented in bold inFIG. 1. Some potential sites of phosphorylation by protein kinase C(PKC) or by calmodulin-dependent protein kinases were identified in thethird intracellular loop and in the carboxyterminal part of thereceptor. The putative phosphorylation sites by PKC or bycalmodulin-dependent protein kinases and PKC are indicated respectivelyby black squares and by open circles in FIG. 1. The four positivelycharged amino acid which have been reported to play a role in the P2Y2receptor activation by ATP and UTP (1) are conserved in the P2Y4sequence: His²⁶², Arg²⁶⁵, Lys²⁸⁹ and Arg²⁹² (FIG. 1). The P2Y4 aminoacid sequence was compared to the chick P2Y1 and the murine P2Y2 aminoacid sequences and to their closest neighbours in the G protein-coupledreceptor family (FIG. 2). The plot was constructed using the multiplesequence alignment program Pileup of the GCG package (26). For eachsequence, the analysis takes into account a segment covering the firstfive putative membrane-spanning domains. It is clear that, from astructural point of view, the newly cloned receptor is more closelyrelated to the human P2Y2 receptor (51% of identity between the completesequences) than to the chick P2Y1 receptor (35%).

[0101] 2. Tissue Distribution of the P2Y4 Receptor

[0102] The tissue distribution of P2Y4 transcripts was investigated byNorthern blotting. A number of rat tissues (heart, brain, liver, testisand kidney) were tested using a human probe at low stringency, but nohybridization signal could be obtained. No P2Y4 transcript could bedetected in the following human cell lines: K562 leukemia cells (FIG.3), HL-60 leukemia cells and SH-SY5Y human neuroblastoma cells. TheNorthern blot was performed with 15 μg of total RNA from human placentaand 4 μg of poly(A)⁺ RNA from K562 cells and from two different humanplacentas. The probe was the human P2Y4 gene fragment amplified by PCR(TM2 to TM7). On the contrary, a strong signal, corresponding to a 1.8kb mRNA, was found in human placenta (FIG. 3).

[0103] 3. Functional Expression of the New P2 Receptor in 1321N1 Cells

[0104] After transfection of the pcDNA3-P2Y4 construction in 1321N1cells, the pool of G418-resistant clones was tested for their functionalresponse (IP3 accumulation) to ATP and UTP. Both nucleotides were foundto be agonists of the P2Y4 receptor, but the response to UTP was morerobust. About 20 transfected clones were then isolated and tested fortheir response to UTP. The clone presenting the highest IP accumulationfactor in response to UTP was selected and used in all subsequentexperiments. Functional characterization of the P2Y₄ receptor wasperformed by determining the accumulation of InsP₃ after 20 minincubation with the agonists in the presence of 10 mM LiCl. We observedthat the response to UTP was biphasic, with a peak reached at 30 s,followed by a more sustained stimulation of lower magnitude (FIG. 4A).With ATP, only that second phase was detectable: its effect becameapparent after 1 min of stimulation only and was stable for at least 20min (FIGS. 4A and B). As for UTP, the stimulation by UDP was biphasic,but it was slightly delayed (FIGS. 4A and B). Inclusion of LiCl hadlittle effect on the initial peak induced by UTP or UDP, but it stronglyenhanced the following plateau phase (FIG. 4B).

[0105] The maximal effect of ATP observed after a 20 min incubationrepresented about 27±9% of that of UTP (mean±S.D. of ten experiments).In order to demonstrate that ATP is able to antagonize the UTP response,incubations of 1321N1 cells were conducted with ATP alone or incombination with UTP. FIG. 5 shows that at high concentration (500 μM ormore), ATP was able to inhibit the effect of UTP, both at 30 s and 20min. At 30 s, the response to UTP 10 μM was fully antagonized by ATP 2mM, corresponding to the fact that ATP has no effect on the human P2Y₄receptor at this early time (panel A). At 20 min, an inhibition of62±11% of the UTP effect (10 μM), corresponding to the differencebetween the UTP and the ATP effects, was observed in the presence of 2mM ATP (mean±S.D. of five independent experiments) (panels B and C). TheATP concentration-inhibition curves were shifted to the right when theUTP concentration was increased, indicating the competitive nature ofthis inhibitory effect (panels A and B). On the other hand, at lowerconcentrations (30-300 μM), ATP enhanced the response to UTP by 29%(range 12-47%, mean of four experiments) (panel B). ADP, which hadalmost no effect per se and did not inhibit the action of UTP,reproduced that enhancement: in the presence of ADP (100 μM), thestimulation by UTP (10 μM) represented 158±15% (mean of threeindependent experiments) of that by UTP alone (data not shown). However,this potentiating effect of ATP and ADP was not specific: indeed theaction of carbachol mediated by muscarinic receptors endogenouslyexpressed in the 1321N1 cells (6) was also increased in the presence ofthese nucleotides. This observation was reproduced with cellstransfected with the recombinant P2Y₄-pcDNA3 plasmid or with the vectoralone and was also obtained with AMP and adenosine (data not shown).

[0106] We compared the concentration-action curves of UTP and UDP on theInsP₃ production for several clones of transfected cells. The study wasmade at two times (FIG. 6): 30 s and 20 min. In the set of experimentsperformed on clone 11 (clone of 1321N1 transfected cells chosen for thepharmacological characterization), UTP appeared to be 10-fold morepotent than UDP after a 20 min incubation and this difference wasreproduced with two other clones (FIG. 6). The EC₅₀ values were 0.3±0.1μM and 3.3±0.6 μM in clone 2, 2.4±0.1 μM and 19.8±4.8 μM in clone 11 and0.3±0.1 μM and 3.2±0.8 μM in clone 21, respectively, for UTP and UDP(mean±S.D. of two independent experiments). At 30 s of incubation, itwas not possible to determine EC₅₀ values because the curves wereclearly shifted to the right, but we can observe that the differencebetween the two agonists potency was even more striking (FIG. 6).Several clones, including clones 2, 11 and 21 were tested in bindingstudies with [α³²P] UTP but no increase in specific binding was observedas compared to the cells transfected with the vector alone (data notshown).

[0107] In view of the time differences observed in FIG. 6, the testingof a range of nucleotides was performed at two times: 30 s and 20 min.As FIG. 7 shows, several agonists were barely or not active at 30 s(UDP, 5BrUTP, dUTP, ITP) whereas they produced a significant effect at20 min. Full concentration-action curves were obtained at 20 min. Therank order of potency was: UTP>UDP=dUTP>5BrUTP>ITP>ATP (FIG. 8). TheEC₅₀ values obtained were the following: EC₅₀UTP=2.5±0.6 μM, EC₅₀UDP=19.5±3.9 μM (mean±S.D. of eight independent experiments), EC₅₀dUTP=20.0±2.3 μM, EC₅₀ 5BrUTP=27.1±1.9 μM and EC₅₀ ITP=32.8±5.4 μM(mean±S.D. of two independent experiments). The approximative EC₅₀ valueobtained for ATP was: 43±12 μM (mean±S.D. of five independentexperiments). The diadenosine polyphosphates also increased the InsP₃production in transfected cells with EC₅₀ between 3 and 7 μM (data notshown), but their maximal effect was only 20-25% of that of UTP, a valueclose to that of ATP (range of four independent experiments) (FIG. 7).UMP, uridine, AMP, adenosine and ATPyS were without any effect (data notshown).

[0108] No specific antagonist is available for any P2Y subtype.Nonetheless, several non-selective antagonists such as suramin, RB2 orPPADS have been tested on P₂ receptors and their relative actions onthese subtypes may constitute a mean to discriminate them (27). So wetested the ability of these three antagonists to inhibit the UTPresponse in the model of the human P2Y₄ receptor. As we can see on FIG.9, PPADS appeared to be the most active antagonist (73±14% inhibition;IC₅₀ around 15 μM (data not shown)), suramin was inactive, and RB-2produced an inhibition of 33±5% of the UTP response (mean+S.D. of twoindependent experiments). FIG. 10 shows the mixed nature of theantagonism by PPADS of the UTP response: it affects both the EC₅₀ valueand the maximal effect of UTP. The EC₅₀ value for UTP in the absence ofPPADS was 3.3±0.6 μM and 12.2±4.5 μM in the presence of 100 μM PPADS(mean±S.D. of two independent experiments).

[0109] The effect of pertussis toxin (20 ng/ml, 18 hours pretreatment)was studied at different times after UTP (100 μM) addition (FIG. 11).The UTP response was clearly inhibited at 30 s (62±5% of inhibition:mean±S.D. of two independent experiments), whereas no significant effectwas observed at 5 and 20 min.

[0110] References

[0111] 1. Erb, L., Garrad, R., Wang, Y., Quinn, T., Turner, J. T., andWeisman, G. A. (1995) J. Biol. Chem. 270, 4185-4188.

[0112] 2. Fredholm, B. B., Abbracchio, M. P., Burnstock, G., Daly, J.W., Harden, T. K., Jacobson, K. A., Leff, P., and Williams, M. (1994)Pharm. Rev. 46, 143-156.

[0113] 3. Valera, S., Hussy, N., Evans, R. J., Adami, N., North, R. A.,Surprenant, A., and Buell, G. (1994) Nature 371, 516-519.

[0114] 4. Brake, A. J., Wagenbach, M. J., and Julius, D. (1994) Nature371, 519-523.

[0115] 5. Webb, T. E., Simon, J., Krishek, B. J., Bateson, A. N., Smart,T. G., King, B. F., Burnstock, G., and Barnard, E. A. (1993) FEBS 324,219-225.

[0116] 6. Filtz, T. N., Li, Q., Boyer, J. L., Nicholas, R. A., andHarden, T. K. (1994) Mol. Pharm. 46, 8-14.

[0117] 7. Henderson, D. J., Elliot, D. G., Smith, G. M., Webb, T. E.,and Dainty, I. A. (1995) Biochem. Biophys. Res. Commun. 212, 648-656.

[0118] 8. Tokoyama, Y., Hara, M., Jones, E. M. C., Fan, Z., and Bell, G.I. (1995) Biochem. Biophys. Res. Commun. 211, 211-218.

[0119] 9. Lustig, K. D., Shiau, A. K., Brake, A. J., and Julius, D.(1993) Proc. Natl. Acad. Sci. 90, 5113-5117.

[0120] 10. Erb, L., Lustig, K. D., Sullivan, D. M., Turner, J. T., andWeisman, G. A. (1993) Proc Natl Acad Sci 90, 10449-10453.

[0121] 11. Rice, W. R., Burton, F. M., and Fiedeldey, D. T. (1995) Am.J. Respir. Cell, Molec. Biol. 12, 27-32.

[0122] 12. Parr, C. E., Sullivan, D. M., Paradiso, A. M., Lazarowski, E.R., Burch, L. H., Olsen, J. C., Erb, L., Weisman, G. A., Boucher, R. C.,and Turner, J. T. (1994) Proc. Natl. Acad. Sci. 91, 3275-3279.

[0123] 13. Barnard, E. A., Burnstock, G., and Webb, T. E. (1994) TiPS15, 67-70.

[0124] 14. Kaplan, M. H., Smith, D. I., and Sundick, R. S. (1993) J.Immun. 151, 628-636.

[0125] 15. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning: A laboratory Manual (Cold Spring Harbor Lab. Press,Plainview, N.Y.).

[0126] 16. Velu, T. J., Beguinot, L., Vass, W. C., Zhang, K., Pastan,I., and Lowry, D. R. (1989) J. Cell. Biochem. 39, 153-166.

[0127] 17. Communi, D., Raspe, E., Pirotton, S., and Boeynaems, J. M.(1995) Circ. Res. 76, 191-198.

[0128] 18. Libert, F., Parmentier, M., Lefort, A., Dinsart, C., VanSande, J., Maenhaut, C., Simons, M. J., Dumont, J. E., and Vassart, G.(1989) Science 244, 569-572.

[0129] 19. Zeng, D., Harrison, J. K., D'Angelo, D. D., Barber, C. M.,Tucker, A. L., Lu, Z., and Lynch, K. R. (1990) Proc. Natl. Acad. Sci.87, 3102-3106.

[0130] 20. Nomura, H., Nielsen, B. W., and Matsushima, K. (1993) Int.Immun. 5, 1239-1249.

[0131] 21. Harrison, J. K., Barber, C. M., and Lynch, K. R. (1994)Neuroscience Letters 169, 85-89.

[0132] 22. Seifert, R. and Schultz, G. (1989) TiPS 10, 365-369.

[0133] 23. Brown, H. A., Lazarowski, E. R., Boucher, R. C., and Harden,T. K. (1991) Mol. Pharm. 40, 648-655.

[0134] 24. O'Connor, S. E., Dainty, I. A., and Leff, P. (1991) TiPS 12,137-141.

[0135] 25. Lazarowski, E. R. and Harden, T. K. (1994) J. Biol. Chem.269, 11830-11836.

[0136] 26. Devereux, J., Haeberli, P. and Smithies 0. A. (1984) NucleicAcids Res. 12, 387-395.

[0137] 27. Motte S., Swillens S. and Boeynaems J. M. (1996) Eur. J.Pharmacol. 307, 201.

[0138] 28. Boyer, J. L., Zohn, I. E., Jacobson, K. A. and Harden, T. K.(1994) Br. J. Pharmacol. 113, 614.

[0139] All patents, patent applications, and published references citedherein are hereby incorporated by reference in their entirety. Whilethis invention has been particularly shown and described with referencesto preferred embodiments thereof, it will be understood by those skilledin the art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1 4 1 1429 DNA Homo sapiens 1 aagggagctt gggtaggggc caggctagcctgagtgcacc cagatgcgct tctgtcagct 60 ctccctagtg cttcaaccac tgctctccctgctctacttt ttttgctcca gctcagggat 120 gggggtgggc agggaaatcc tgccaccctcacttctcccc ttcccatctc caggggggcc 180 atggccagta cagagtcctc cctgttgagatccctaggcc tcagcccagg tcctggcagc 240 agtgaggtgg agctggactg ttggtttgatgaggatttca agttcatcct gctgcctgtg 300 agctatgcag ttgtctttgt gctgggcttgggccttaacg ccccaaccct atggctcttc 360 atcttccgcc tccgaccctg ggatgcaacggccacctaca tgttccacct ggcattgtca 420 gacaccttgt atgtgctgtc gctgcccaccctcatctact attatgcagc ccacaaccac 480 tggccctttg gcactgagat ctgcaagttcgtccgctttc ttttctattg gaacctctac 540 tgcagtgtcc ttttcctcac ctgcatcagcgtgcaccgct acctgggcat ctgccaccca 600 cttcgggcac tacgctgggg ccgccctcgcctcgcaggcc ttctctgcct ggcagtttgg 660 ttggtcgtag ccggctgcct cgtgcccaacctgttctttg tcacaaccag caacaaaggg 720 accaccgtcc tgtgccatga caccactcggcctgaagagt ttgaccacta tgtgcacttc 780 agctcggcgg tcatggggct gctctttggcgtgccctgcc tggtcactct tgtttgctat 840 ggactcatgg ctcgtcgcct gtatcagcccttgccaggct ctgcacagtc gtcttctcgc 900 ctccgctctc tccgcaccat agctgtggtgctgactgtct ttgctgtctg cttcgtgcct 960 ttccacatca cccgcaccat ttactacctggccaggctgt tggaagctga ctgccgagta 1020 ctgaacattg tcaacgtggt ctataaagtgactcggcccc tggccagtgc caacagctgc 1080 ctggatcctg tgctctactt gctcactggggacaaatatc gacgtcagct ccgtcagctc 1140 tgtggtggtg gcaagcccca gccccgcacggctgcctctt ccctggcact agtgtccctg 1200 cctgaggata gcagctgcag gtgggcggccaccccccagg acagtagctg ctctactcct 1260 agggcagata gattgtaaca cgggaagccggcaagtgaga gaaaagggga tgagtgcagg 1320 gcagaggtga gggaacccaa tagtgatacctggtaaggtg cttcttcctc ttttccaggc 1380 tctggagaga agccctcacc ctgagggttgccacggaggc agggatatc 1429 2 365 PRT Homo sapiens 2 Met Ala Ser Thr GluSer Ser Leu Leu Arg Ser Leu Gly Leu Ser Pro 1 5 10 15 Gly Pro Gly SerSer Glu Val Glu Leu Asp Cys Trp Phe Asp Glu Asp 20 25 30 Phe Lys Phe IleLeu Leu Pro Val Ser Tyr Ala Val Val Phe Val Leu 35 40 45 Gly Leu Gly LeuAsn Ala Pro Thr Leu Trp Leu Phe Ile Phe Arg Leu 50 55 60 Arg Pro Trp AspAla Thr Ala Thr Tyr Met Phe His Leu Ala Leu Ser 65 70 75 80 Asp Thr LeuTyr Val Leu Ser Leu Pro Thr Leu Ile Tyr Tyr Tyr Ala 85 90 95 Ala His AsnHis Trp Pro Phe Gly Thr Glu Ile Cys Lys Phe Val Arg 100 105 110 Phe LeuPhe Tyr Trp Asn Leu Tyr Cys Ser Val Leu Phe Leu Thr Cys 115 120 125 IleSer Val His Arg Tyr Leu Gly Ile Cys His Pro Leu Arg Ala Leu 130 135 140Arg Trp Gly Arg Pro Arg Leu Ala Gly Leu Leu Cys Leu Ala Val Trp 145 150155 160 Leu Val Val Ala Gly Cys Leu Val Pro Asn Leu Phe Phe Val Thr Thr165 170 175 Ser Asn Lys Gly Thr Thr Val Leu Cys His Asp Thr Thr Arg ProGlu 180 185 190 Glu Phe Asp His Tyr Val His Phe Ser Ser Ala Val Met GlyLeu Leu 195 200 205 Phe Gly Val Pro Cys Leu Val Thr Leu Val Cys Tyr GlyLeu Met Ala 210 215 220 Arg Arg Leu Tyr Gln Pro Leu Pro Gly Ser Ala GlnSer Ser Ser Arg 225 230 235 240 Leu Arg Ser Leu Arg Thr Ile Ala Val ValLeu Thr Val Phe Ala Val 245 250 255 Cys Phe Val Pro Phe His Ile Thr ArgThr Ile Tyr Tyr Leu Ala Arg 260 265 270 Leu Leu Glu Ala Asp Cys Arg ValLeu Asn Ile Val Asn Val Val Tyr 275 280 285 Lys Val Thr Arg Pro Leu AlaSer Ala Asn Ser Cys Leu Asp Pro Val 290 295 300 Leu Tyr Leu Leu Thr GlyAsp Lys Tyr Arg Arg Gln Leu Arg Gln Leu 305 310 315 320 Cys Gly Gly GlyLys Pro Gln Pro Arg Thr Ala Ala Ser Ser Leu Ala 325 330 335 Leu Val SerLeu Pro Glu Asp Ser Ser Cys Arg Trp Ala Ala Thr Pro 340 345 350 Gln AspSer Ser Cys Ser Thr Pro Arg Ala Asp Arg Leu 355 360 365 3 35 DNAartificial sequence Primer for the second transmembrane region of humanpyrimidine receptor 3 cagatctaga tactatgttc tacactctta cgtgc 35 4 35 DNAartificial sequence primer for seventh transmembrane region of humanpyrimidine receptor 4 tcttaagctt ggagtcacgt acgagcaagc tagtt 35

We claim:
 1. An isolated antibody which specifically binds to a proteinreceptor, wherein said receptor comprises the amino acid sequence shownin SEQ ID NO:2.
 2. The antibody of claim 1, wherein said antibody is anagonist of said receptor.
 3. The antibody of claim 1, wherein saidantibody is an antagonist of said receptor.
 4. The antibody of any oneof claims 1, 2 or 3, wherein said antibody is a monoclonal antibody. 5.The monoclonal antibody of claim 4, wherein said monoclonal antibody isdirected to an epitope of said receptor, wherein said epitope is presenton the surface of a cell expressing said receptor.
 6. A pharmaceuticalcomposition comprising the antibody of claims 1, 2 or 3, and also apharmaceutically acceptable carrier.
 7. The pharmaceutical compositionof claim 6, wherein said antibody is a monoclonal antibody.
 8. A methodfor determining whether an antibody can specifically bind to a receptor,wherein said receptor comprises the amino acid sequence shown in SEQ IDNO:2, comprising the following steps: i) contacting a cell whichexpresses said receptor with the antibody, wherein said cell istransfected with an expression vector comprising the nucleic acidmolecule encoding said receptor, and wherein said contacting is underconditions permitting binding of said antibody to said receptor, and ii)detecting the presence or absence of the antibody bound specifically tosaid receptor, wherein the presence of the antibody indicates that theantibody can specifically bind to said receptor.
 9. A method fordetermining whether an antibody can specifically bind to a receptor,wherein said receptor comprises the amino acid sequence shown in SEQ IDNO:2, comprising the following steps: i) preparing a cell extract fromcells transfected with an expression vector comprising the nucleic acidmolecule encoding said receptor, ii) isolating a membrane fraction fromthe cell extract, iii) contacting the antibody with the membranefraction under conditions permitting binding of the antibody to saidreceptor, and iv) detecting the presence or absence of the antibodybound specifically to said receptor, wherein the presence of theantibody indicates that the antibody can specifically bind to saidreceptor.
 10. A method for determining whether an antibody is an agonistof a receptor, wherein said receptor comprises the amino acid sequenceshown in SEQ ID NO:2, comprising the following steps: i) contacting acell, which expresses said receptor, with the antibody, wherein saidcell is transfected with an expression vector comprising the nucleicacid molecule encoding said receptor, wherein said contacting is underconditions permitting the activation of a functional receptor responsefrom the antibody, and ii) measuring the receptor activity by means of abio-assay, wherein an increase of bioactivity indicates that theantibody is a receptor agonist.
 11. A method for determining whether anantibody is an agonist of a receptor, wherein said receptor comprisesthe amino acid sequence shown in SEQ ID NO:2, comprising the followingsteps: i) preparing a cell extract from cells which express saidreceptor, wherein said cell is transfected with an expression vectorcomprising the nucleic acid molecule encoding said receptor, ii)isolating a membrane fraction from the cell extract, iii) contacting themembrane fraction with the antibody under conditions permitting theactivation of a functional receptor response, and iv) measuring thereceptor activity by means of a bio-assay, wherein an increase ofbioactivity indicates that the antibody is a receptor agonist.
 12. Amethod for determining whether an antibody is an antagonist of areceptor, wherein said receptor comprises the amino acid sequence shownin SEQ ID NO:2, comprising the following steps: i) contacting a cellwhich expresses said receptor, with the antibody in the presence of aknown receptor agonist, wherein said cell is transfected with anexpression vector comprising a nucleic acid molecule encoding saidreceptor, wherein said contacting is under conditions permitting theactivation of a functional receptor response, and ii) measuring theactivity of said receptor by means of a bio-assay, wherein a decrease insaid activity indicates that the antibody is a receptor antagonist. 13.A method for determining whether an antibody is an antagonist of areceptor, wherein said receptor comprises the amino acid sequence shownin SEQ ID NO:2, comprising the following steps: i) preparing a cellextract from cells which express said receptor, wherein said cells aretransfected with an expression vector comprising the nucleic acidmolecule encoding said receptor, ii) isolating a membrane fraction fromthe cell extract, iii) contacting the membrane fraction with theantibody in the presence of a known receptor agonist, wherein saidcontacting occurs under conditions permitting the activation of afunctional receptor response, and iv) measuring the activity of saidreceptor by means of a bio-assay, wherein a decrease in said activityindicates that the antibody is a receptor antagonist.
 14. The methodaccording to any one of claims 10, 11, 12 and 13, wherein said bio-assaymeasures a modification in a second messenger concentration or amodification in the cellular metabolism.
 15. The method according toclaim 14, wherein said bio-assay comprises a measurement ofintra-cellular cAMP, intra-cellular inositol phosphate, intra-cellulardiacylglycerol concentration, or intra-cellular calcium mobilization.16. The method according to any one of claims 10, 11, 12 and 13, whereinthe cell is a mammalian cell.
 17. The method according to claim 16,wherein said mammalian cell is selected from the group consisting ofCOS-7 cells, CHO cells, LM(tk-) Cells, NIH-3T3 cells or 1321N1 cells.18. A method of detecting the presence of a receptor on the surface of acell, wherein said receptor comprises the amino acid sequence shown inSEQ ID NO:2, wherein said method comprises contacting the cell with theantibody of claim 1 under conditions permitting the binding of theantibody to the receptor, and detecting the presence of the antibodybound to the cell, thereby detecting the presence of the receptor on thesurface of the cell.
 19. A method for diagnosing a predisposition to adisorder associated with the activity of a specific allele of areceptor, wherein said receptor comprises the amino acid sequence shownin SEQ ID NO:2, wherein said method comprises: a) obtaining nucleic acidmolecules of subjects suffering from said disorder; b) performing arestriction digest of said nucleic acid molecules with a panel ofrestriction enzymes; c) electrophoretically separating the resultingnucleic acid fragments on a sized gel; d) contacting the resulting gelwith a nucleic acid probe capable of specifically hybridizing to saidnucleic acid molecules and labeled with a detectable marker; e)detecting labeled bands which have hybridized to the said nucleic acidmolecule labeled with a detectable marker to create a unique bandpattern specific to subjects suffering from said disorder; f) preparingnucleic acid molecules obtained for diagnosis from subjects without apredisposition for the disorder by steps a-e; and g) comparing theunique band pattern specific to the nucleic acid molecule of subjectssuffering from the disorder from step e and the nucleic acid moleculeobtained for diagnosis from step f) to determine whether the patternsare the same or different and to diagnose thereby predisposition to thedisorder if the pattern are the same.